Molecular fluorescence methods ease determination of cell line quality
Human embryonic stem cells promise to provide novel treatments for various diseases and conditions, including ones that currently have no cure, such as paralysis and neurodegenerative disorders. The reliability of these treatments depends upon the quality of the stem cell lines maintained in repositories. In particular, chromosomal changes can occur in these cell lines. Conventional cytogenetic techniques, in which chromosomes are harvested from cells in metaphase, can reveal these abnormalities, as can state-of-the-art molecular fluorescence methods. Each process has unique advantages and limitations, according to a review written by researchers from the Andalusian Stem Cell Bank and from the Hospital Virgen de las Nieves, both in Granada, Spain.
Conventional cytogenetic methods are regarded as the gold standard for detecting chromosomal changes because they enable screening of the entire genome. However, they cannot be automated. Furthermore, they require cells to divide, and inducing basic cell division can be difficult and time-consuming, let alone creating cell divisions with the chromosomes clearly defined for analysis. In contrast, methods based on fluorescence in situ hybridization can be automated and do not require cell division, so they can be performed relatively quickly, but they cannot analyze all of the chromosomes at once.
A technique based on fluorescence in situ hybridization, called comparative genomic hybridization, can analyze all of the chromosomes simultaneously, although it can miss certain abnormalities. It requires a reference control DNA, which is labeled with the red fluorophore rhodamine. DNA is extracted from the cells from the cell line and is labeled with the green fluorophore fluorescein. The control and cell DNA are mixed together, and they hybridize. Normal DNA will appear yellow, whereas lost genetic material will appear red, and gained material will appear green. The red:green ratio is analyzed by software.
The researchers concluded that comparative genomic hybridization, fluorescence in situ hybridization and conventional cytogenetic methods are complementary. (Cell Biology International, September 2007, pp. 861-869.)
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